Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.
The main results reproduced: recomputed values matched the published ones within tolerance.
- ✓Same input data as the authors
- 🟡Reported values were only indirectly comparable
- 🟡A deviation arose in the data or preprocessing
- 🟡A deviation was attributed to the published material
- 🟡Reported values were not (fully) derivable from the shared data
- 🟡The deviation was non-trivial in magnitude
- 🟡The central claim did not (fully) hold under reproduction
- 🟡Overall, the reproduction showed a material discrepancy
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
DESCRIBED WELL ENOUGH AND REPRODUCED ~1:1 (independent re-run, «job»). GSE162210 = bulk PBMC Affymetrix Clariom S microarray, 20 samples (4 donors x 5 groups: Mock/IAV/IAV+IA/IAV+DI/IAV+4OI); N reported(20)==N observed(20), complete & balanced, grade A. Pipeline = oligo RMA -> PCAtools::pca (the paper's own code link kevinblighe/PCAtools = P16 third-party tool) -> limma one-way ANOVA + contrasts -> limma::goana GO, ran on «our HPC» SLURM («job», 7m21s incl. fresh conda env build + preprocessCore source rebuild, node n094). The RMA expression matrix is BYTE-IDENTICAL (sha256 063cdcbf...) to the earlier run -> fully deterministic. All four in-scope GSE162210 results reproduce: (C1) PCA recovers S18 exactly -- 4OI is the only discernible far-right group, exactly one outlying DI sample (GSM4946691, PC2=132.9), two deviating itaconate samples; (C2) 4OI drives by far the most DEGs (5562 vs ~1500 for IA/DI); (C3) HMOX1 strongly induced by DI (logFC +2.75, adj.P 4.6e-09); (C4) 4OI/DI DEGs enrich antiviral/interferon/virus GO terms at FDR<0.02. NOT attempted (out of scope): dTHP-1 microarray, scRNA-seq, all wet-lab. Gotchas confirmed: rma() takes no target= for Clariom S; env auto-resolved preprocessCore 1.68.0 and RMA ran cleanly under SLURM (no pthread crash this run); point TMPDIR/CONDA_PKGS_DIRS at «infra» (front1 /tmp tiny). All grades PROVISIONAL pending human sign-off; no fabrication concern (every value derivable from the deposited CELs).
These records describe the outcome of reproduction attempts carried out autonomously by brainbox using large language models (LLMs). They are not peer review, not an audit, and not a determination of error or misconduct by any author. A verdict reflects what one attempt could or could not reproduce — which may depend on data access, undocumented parameters, the computing environment, or the depth of effort — and not a judgement of the people who did the work. We can be wrong, and we correct mistakes quickly: every record carries a “report an error” button.
Assessment versions
Every reproduction run is kept as an immutable version — anchored to the data as it stood, with a tamper-evident chain hash. A rerun (e.g. after an author updates a deposit) adds a new version; the previous one stays on record.
-
v1 current initial assessment Score 95assessed: 2026-06-21 ⛓ 7b7330c61477
✎ I am an author of this paper
Updated or fixed a deposit, or is there an erratum? Ask us to re-run the metrics. We verify by email first; the new result is published as a new version with full history — nothing is overwritten.
Provenance — full disclosure
When this reproduction was carried out, which methodology version was used, and by whom — so the record can be audited and checked independently.
- Reproduced
- 2026-06-25
- Rubric version
- v1.0
- Assessed by
-
🤖 AI curator · claude (ai-curator room) · v1.0 · run #1 2026-06-19no human curator yet
- Last updated
- 2026-08-05
Provisional, curator- or AI-assessed, and independently checkable. A reproduction outcome states what one attempt could reproduce — not a judgement of the authors.
Deep full-text extraction
Model: sonnetThe paper tests whether the endogenous ACOD1/itaconate axis modulates host inflammatory and antiviral responses to influenza A virus (IAV) infection, and whether exogenous application of itaconate and its derivatives (dimethyl-itaconate, 4-octyl-itaconate) can reduce IAV-induced inflammation without compromising (and potentially enhancing) antiviral control.
- ★ Acod1 expression and itaconate synthesis are induced in mouse lung during IAV infection and correlate with inflammation and disease severity finding
- ★ Acod1-/- mice show greater weight loss, mortality, and pulmonary inflammation than wild-type mice during IAV infection finding
- ★ Dimethyl-itaconate (DI) treatment reduces pulmonary inflammation and mortality in IAV-infected mice finding
- ★ Itaconate, DI, and 4OI reverse infection-triggered interferon responses and modulate inflammation in human cell lines, PBMCs, and lung tissue finding
- ★ All three itaconates reduce ROS levels and STAT1 phosphorylation; AKT phosphorylation is reduced by 4OI/DI but increased by unmodified itaconate finding
- ★ Monocytes are the main target of IAV infection and the exclusive source of ACOD1 mRNA in peripheral blood (single-cell RNA-seq) finding
- ★ Viral replication is not increased despite strongly repressed IFN responses; 4OI inhibits viral transcription in PBMCs, and itaconate/4OI reduce viral titers in A549 cells (4OI>Ita>DI) finding
- ★ IAV infection induces ACOD1 mRNA expression in human PBMCs and macrophages, with viral transcription highest in M2-differentiated macrophages finding
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| bulk RNA-seq | mouse lung (C57BL/6J vs DBA/2J) | IAV infection (PR8M) | Acod1, Tnfaip3, Hmox1/2 mRNA expression over time course | — |
| metabolomics (itaconate quantification) | mouse lung (C57BL/6, Acod1+/+, +/-, -/-) | IAV infection / Acod1 genotype | pulmonary itaconate concentration | — |
| histopathology (H&E staining, semi-quantitative scoring) | mouse lung (C57BL/6N, Acod1 genotypes) | IAV infection / Acod1 genotype | pulmonary inflammation score | — |
| in vivo weight loss and survival assay | female C57BL/6N mice (Acod1+/+, +/-, -/-) | IAV infection / Acod1 genotype; DI treatment | weight loss (%), survival/mortality | — |
| mRNA expression (qPCR/microarray) | human PBMCs, PBMC-derived M1/M2 macrophages, dTHP-1 cells | IAV infection | ACOD1 mRNA levels, viral transcription | — |
| single-cell RNA sequencing | human PBMCs | IAV infection; DI treatment | cell-type-specific gene expression (IFN response genes, ACOD1) | — |
| Western blot | human cells / macrophages | IAV infection + itaconate/DI/4OI treatment | STAT1 and AKT phosphorylation, ROS levels | — |
| viral titer / plaque assay | A549 human lung epithelial cells | IAV infection + itaconate/DI/4OI treatment | infectious viral titer, viral transcription | — |
- ▲ Acod1 was the 4th most highly upregulated pulmonary mRNA 48h post-IAV infection in C57BL/6J mice 4th most upregulated transcript
- ▲ Itaconate concentrations were higher in IAV-infected DBA/2J than C57BL/6J mouse lungs and correlated with HA and Acod1 mRNA levels
- ▼ Median survival after day 7 was significantly lower in Acod1-/- than Acod1+/+ mice p=0.018
- ▲ Weight loss and mortality were greater in Acod1-/- than Acod1+/+ mice, with largest differences around day 8
- ▲ Pulmonary inflammation was greatest in Acod1-/- and intermediate in Acod1+/- mice
- ▲ Viral transcription was highest in M2-differentiated macrophages compared with M1 and dTHP-1 cells
- ▼ 4OI strongly inhibited viral transcription in PBMCs
- ▼ Itaconate and 4OI reduced viral titers in A549 cells (4OI>Ita>DI) while viral transcription was unaffected
- pvalue p=0.018 (Median survival after day 7, Acod1-/- vs Acod1+/+ mice (Mann-Whitney U test))
- count n=36 Acod1+/+, 27 Acod1+/-, 4 Acod1-/- (Mice used for pulmonary itaconate quantification)
- count n=9 Acod1+/+, 10 Acod1+/-, 9 Acod1-/- (Female mice used in weight loss/survival/histopathology experiment)
- count n=3 per strain and time point (RNAseq analysis of mouse lung gene expression (DBA/2J vs C57BL/6J))
- fold_change 4th most highly upregulated transcript (Acod1 mRNA ranking in C57BL/6J mouse lung 48h post IAV infection)
- other 4OI>Ita>DI (Relative potency ranking for reducing viral titers in A549 cells)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
The paper investigated endogenous and exogenous itaconate effects on influenza A virus (IAV) infection using mouse knockout models, human cell lines, PBMCs, explanted lung tissue, and single-cell RNA sequencing. Mouse genotype comparisons (Acod1+/+, +/-, -/-) were analyzed with unpaired t-tests, two-way ANOVA with Tukey post-hoc, and a Mann-Whitney U for survival; bulk and single-cell RNA-seq data were analyzed with pairwise t-tests corrected by Benjamini-Hochberg FDR. Results were predominantly reported with threshold-based significance markers (*, **, ***) rather than exact p-values, and no effect sizes or dispersion measures were described in the available text.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| Pairwise t-test with Benjamini-Hochberg multiple testing correction | Fig 1A — strain comparison (DBA/2J vs C57BL/6J) of pulmonary Acod1, Tnfaip3, Hmox1/2 mRNA expression across time points (reanalysis of published RNAseq data) | n=3 per strain per time point | not stated |
| Unpaired t-test | Fig 1C — pulmonary itaconate levels across Acod1 genotypes (+/+, +/-, -/-) in IAV-infected mice | n=36 Acod1+/+, 27 Acod1+/-, 4 Acod1-/- | not stated |
| Unpaired t-test | Fig 1D — weight loss trajectories in female Acod1+/+, +/-, and -/- C57BL/6N mice during IAV infection | n=9 Acod1+/+, 10 Acod1+/-, 9 Acod1-/- | not stated |
| Mann-Whitney U test | Fig 1E — survival comparison across Acod1 genotypes in the same mouse experiment; reported p=0.018 for Acod1-/- vs Acod1+/+ | n=9 Acod1+/+, 10 Acod1+/-, 9 Acod1-/- | not stated |
| Two-way ANOVA with Tukey's multiple comparisons post-hoc test | Fig 1F — semi-quantitative histopathological inflammation scores across Acod1 genotypes and time points (mid/late infection) | n=9 Acod1+/+, 10 Acod1+/-, 9 Acod1-/- (all female) | not stated |
-
Survival differences across the three Acod1 genotypes were assessed with a Mann-Whitney U test (Fig 1E), with the result reported as median survival after day 7.↳ Could also: Kaplan-Meier estimator with a log-rank (Mantel-Cox) test, optionally extended to a Cox proportional-hazards model for multi-group comparison. — Log-rank and Cox models are designed for time-to-event data with right-censored observations (here, animals euthanized at the 20% weight-loss threshold at varying days), explicitly account for when events occur across the full follow-up period, and are the conventional standard for survival analysis in animal studies.
-
Itaconate concentrations and weight loss across three genotypes (Acod1+/+, +/-, -/-) were each tested with pairwise unpaired t-tests (Figs 1C, 1D).↳ Could also: One-way ANOVA (or Kruskal-Wallis if normality is not assumed) followed by a single post-hoc correction (e.g., Tukey HSD or Dunn's test) for the three pairwise contrasts. — A single omnibus test followed by corrected pairwise contrasts jointly controls the family-wise error rate across the three comparisons; multiple separate t-tests without correction inflate the Type I error rate proportionally to the number of tests.
-
Weight loss over the infection time course was compared between genotypes using unpaired t-tests at individual time points (Fig 1D).↳ Could also: A linear mixed-effects model or repeated-measures ANOVA with a genotype × time interaction term. — Repeated measurements on the same animals across days are correlated; mixed-effects or repeated-measures models account for within-subject correlation, can test the overall trajectory rather than isolated time points, and may improve statistical power.
-
p-values are predominantly reported as threshold brackets (* <0.05, ** <0.01, *** <0.001) rather than exact values (Figs 1A, 1C, 1D, 1F).↳ Could also: Report the exact p-value for each comparison (e.g., p=0.032 rather than *). — Exact p-values convey the continuous strength of evidence, allow readers to apply their own thresholds, and facilitate downstream systematic review, meta-analysis, and replication assessment; reporting only brackets discards information already computed by the software.
-
No standardized effect size measures (e.g., Cohen's d, eta-squared, rank-biserial correlation) are reported alongside significance tests.↳ Could also: Report a standardized effect size with each primary comparison. — Effect sizes quantify the magnitude of a difference independently of sample size, allowing readers to judge biological or clinical importance and to power future studies; a statistically significant result in a small experiment may correspond to a small or large biological effect, which p-values alone cannot distinguish.
-
Continuous outcome data (e.g., itaconate concentrations, gene expression) are presented without reported dispersion measures (SD, SEM, or CI) in the text.↳ Could also: Accompany means or medians with SD or 95% CI in figures and text. — SD reflects the variability of observations in the sample, while 95% CI reflects uncertainty in the estimated mean; both convey information that p-values and significance markers alone do not, and are especially informative when group sizes are small or unequal (here ranging from n=4 to n=36).
What was reproduced
The exact results taken into scope, with each reported value next to the value our attempt produced.
Scope — PMID 35025971 (Itaconate & IAV; PLoS Pathog 2022)
DOI 10.1371/journal.ppat.1010219 · PMCID PMC8846506 Code link (brief): https://github.com/kevinblighe/PCAtools (a third-party Bioconductor PCA package — P16 case: applying it to the paper's own data is a valid reproduction.) Data (brief): GEO GSE162210.
What GSE162210 actually is
GEO series GSE162210 = the bulk PBMC microarray (Affymetrix Clariom S Human, GPL23159), 20 samples = 4 donors × 5 groups: Mock(uninfected) / IAV / IAV+IA(itaconic acid) / IAV+DI(dimethyl itaconate) / IAV+4OI(4-octyl itaconate). Per Methods (Fig 8C legend): "Microarray analysis of PBMC RNA from four of the donors… RNA pooled from the three replicates of each donor, resulting in 4 samples per group." GSM4946675–GSM4946694. Supplementary: GSE162210_RAW.tar (20 × *.CEL.gz).
Normalization stated: raw .CEL → TAC4.0.2 → RMA. DEGs by limma + one-way ANOVA.
IN SCOPE (pipeline-derived, from GSE162210)
The paper's GSE162210-derived computational results are concentrated in Fig 8C, S18, S19:
| id | result | paper loc | pipeline |
|---|---|---|---|
| C1 | PCA of bulk PBMC transcriptomes: 4OI (all 4) forms a clearly discernible group; one outlying DI sample and two outlying itaconate samples | S18 Fig + Results §"Anti-inflammatory effects…PBMCs" | RMA (oligo) → PCAtools::pca/biplot |
| C2 | "Most significant transcriptome changes driven by 4OI, with major effects on many genes not regulated by IAV/itaconate/DI" | S19 Fig + same §; Methods (limma) | RMA → limma one-way ANOVA, DEG p<0.05 |FC|>1.5 |
| C3 | HMOX1 induced by DI in IAV-infected samples | Results §PBMC | RMA → per-gene contrast DI-IAV vs IAV |
| C4 | GO enrichment of itaconate/DI/4OI effects on IAV-infected PBMCs (DEGs p<0.05 |FC|>1.5; terms FDR<0.02) | Fig 8C legend | DEG → GO/enrichment (harder, attempt last) |
OUT OF SCOPE (not GSE162210 / wet-lab / no code / separate accession)
- All RT-qPCR (ACOD1/TNFAIP3/HA/CXCL10…), ELISA/multiplex cytokines, FACS, ROS, P-STAT1/P-AKT western blots, viral titers — wet-lab (Figs 1,2,4,8A-B,11, Table…).
- Mouse Acod1-/- experiments (Fig… in vivo) — wet-lab.
- dTHP-1 microarray (Fig 3D-I, Venn/volcano, n=3) — a different microarray deposit (NOT GSE162210); no accession given in brief → not attempted here.
- scRNA-seq (Fig 9-10; Seurat V3.1 + DESeq2; 6456 cells) — different data deposit, no accession in brief, no code shipped → out of scope (would be its own RU).
- S17 (reanalysis of an external whole-blood influenza dataset) — external dataset.
Plan
- Download GSE162210_RAW.tar (20 CEL.gz) → «infra» (front1).
- «our HPC» SLURM: oligo RMA (target=core, pd.clariom.s.human) → 20-sample expr matrix.
- PCAtools::pca + biplot colored by group → compare clustering to S18 (C1).
- limma one-way ANOVA across 5 groups + pairwise contrasts; DEG p<0.05 |FC|>1.5; compare 4OI-dominance (C2) and HMOX1 (C3).
- (stretch) GO enrichment of infected-vs-treated DEGs (C4).
Assessments & scoring basis
Each contributor’s verdict, the per-question basis, and the auditable, itemised worksheet behind it.
An automated assessment. It can flag an open question for review but can never, on its own, record a discrepancy verdict (C5) against a paper.
Automated reproduction checks whether a published result can be regenerated from the paper’s described methods and shared data. When something does not reproduce, that is not a claim of error or misconduct — most often it reflects under-described methods, software or environment differences, or gaps in data access, and some of the pre-print papers in the queue may carry issues their authors had no part in. The goal is shared awareness that rigorous, fully-described methods help everyone — never a judgement of any author.
Are you an author? We would genuinely like to hear from you — to clarify the record, add data or code, re-run the pipeline after an accession update, and publish your response right next to the assessment. Everything here is open and auditable.
🚩 Report an error in this record
Spotted something wrong — a verdict you’d contest, a data or value error, or a private detail that slipped through? Tell us, with a short justification. Authors and readers are equally welcome to write in; we review every report.
Prefer email, or the form below not working? Contact us at support@doesitreproduce.com.
Reproduction footprint
claude-opus-4-8Measured resources invested to assess this paper — sanitised (machine class only, no job ids/paths). Compute = HPC accounting (SLURM); tokens = the AI agent's session.